RESEARCH ARTICLE Almeidea A. St.-Hil. Belongs to Conchocarpus J.C. Mikan (Galipeinae, Rutaceae): Evidence from Morphological and Molecular Data, with a First Analysis of Subtribe Galipeinae Carla Poleselli Bruniera1, Jacquelyn A. Kallunki2, Milton Groppo3* 1 Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, São Paulo, a11111 Brazil, 2 The New York Botanical Garden, Bronx, New York, United States of America, 3 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil * [email protected] OPEN ACCESS Abstract Citation: Poleselli Bruniera C, Kallunki JA, Groppo M Subtribe Galipeinae (tribe Galipeeae, subfamily Rutoideae) is the most diverse group of (2015) Almeidea A. St.-Hil. Belongs to Conchocarpus Neotropical Rutaceae, with 28 genera and approximately 130 species. One of its genera is J.C. Mikan (Galipeinae, Rutaceae): Evidence from Almeidea, whose species are morphologically similar to those of the genus Conchocarpus. Morphological and Molecular Data, with a First Analysis of Subtribe Galipeinae. PLoS ONE 10(5): Species of Almeidea occur in the Atlantic Rain Forest of Eastern Brazil, with one species e0125650. doi:10.1371/journal.pone.0125650 (Almeidea rubra) also present in Bolivia. The objective of this study was to perform a phylo- Academic Editor: Zhong-Jian Liu, The National genetic analysis of Almeidea, using a broader sampling of Galipeinae and other Neotropical Orchid Conservation Center of China; The Orchid Rutaceae, the first such study focused on this subtribe. To achieve this objective, morpho- Conservation & Research Center of Shenzhen, logical data and molecular data from the nuclear markers ITS-1 and ITS-2 and the plastid CHINA markers trnL-trnF and rps16 were obtained. Representatives of eight genera of Galipeinae Received: November 17, 2014 and three genera of Pilocarpinae (included also in Galipeeae) and Hortia (closely related to Accepted: March 17, 2015 Galipeeae) were used. Five species of Almeidea and seven of Conchocarpus were includ- Published: May 7, 2015 ed, given the morphological proximity between these two genera. Individual (for each mo- lecular marker) and combined phylogenetic analyses were made, using parsimony and Copyright: © 2015 Poleselli Bruniera et al. This is an open access article distributed under the terms of the Bayesian inference as optimization criteria. Results showed Galipeinae as monophyletic, Creative Commons Attribution License, which permits with the species of Almeidea also monophyletic (supported by the presence of pantocolpo- unrestricted use, distribution, and reproduction in any rate pollen) and nested in a clade with a group of species of Conchocarpus, a non-mono- medium, provided the original author and source are phyletic group. Additionally, C. concinnus appeared in a group with Andreadoxa, credited. Erythrochiton, and Neoraputia, other members of Galipeinae. As a result, Conchocarpus Data Availability Statement: All relevant data are would be monophyletic only with the exclusion of a group of species related to C. concinnus within the paper and its Supporting Information files. and with the inclusion of all species of Almeidea with the group of species of Conchocarpus Funding: The Fundação de Amparo a Pesquisa no that includes its type species, C. macrophyllus. Thus, species of Almeidea are transferred Estado de São Paulo (FAPESP) granted CPB (funding number 2007/06336-0) and MG (funding to Conchocarpus, and the new combinations are made here. number 2006/03170-0 and 2011/10446-0). The Conselho Nacional de Pesquisa (CNPq) granted MG with Productivity in Research Grant (funding number 309994/2012-8). The Universidade de São Paulo provided the facilities. The funders had no role in PLOS ONE | DOI:10.1371/journal.pone.0125650 May 7, 2015 1/20 Almeidea Belongs to Conchocarpus and a First Analysis of Galipeinae study design, data collection and analysis, decision to Introduction publish, or preparation of the manuscript. Rutaceae is a large, predominantly tropical and subtropical family, consisting of 150–164 genera Competing Interests: The authors have declared and 1500–2000 species, with three main centers of diversity: Tropical America, southern Africa, that no competing interests exist. and Australia [1–5]. The family has long been economically important for edible fruits (especial- ly Citrus, the oranges, lemons, tangerines, etc.), aromatic oils (Boronia and Ruta), drugs (e.g., Pilocarpus, source of pilocarpine, used in the treatment of glaucoma), and bitter beverages used to treat fevers (Angostura, Galipea). Many species are used as timbers (Flindersia, Zanthoxylum, Balfourodendron, Euxylophora), and more recently, the antimicrobial and antifungal properties of rutaceous compounds have being exploited and proven to be medicinally useful (e.g., [6–8]). Given their great diversity in morphological characteristics that include a variety of habits, flow- ers, and fruits, allied with a broad geographic distribution, Rutaceae have been traditionally di- vided into subfamilies, tribes, and subtribes, following the classifications of Engler ([9–11], see [12, 13] for a detailed discussion of the these groups). New subfamilial realignments have been recently published, and number of subfamilies varies from two to four [5, 14, 15]. Within the subfamily Rutoideae, the largest of the subfamilies sensu [14], the tribe Galipeeae comprise two subtribes, the Pilocarpinae and the Galipeinae, the latter being by far the most diverse group of Neotropical Rutaceae, with 28 genera and approximately 130 species [13]. Most of the Galipei- nae occur in the Brazilian Atlantic Rain Forest, with some groups reaching the Guianas, the Andes, and Central America [13], mostly in the understory of moist forests [3]. Galipeinae encompass Neotropical Rutaceae with flowers mostly zygomorphic, a more or less tubular corolla, union of the filaments to a corolla tube, reduction of fertile stamens from five to two, basally appendaged anthers, and plicate cotyledons [10, 16](seeFig 1 for some representatives); however, there are exceptions to all the morphological characteristics cited above. Additionally, base chromosome number [17] and pollen morphology [18] are very di- verse in this group. Galipeinae are taxonomically heterogeneous, including 10 monospecific genera (e.g., Adis- canthus, Andreadoxa, Desmotes, Euxylophora, Lubaria), as well as polymorphic genera (e.g. Conchocarpus with 48 species [16, 19]), with most of them occurring in the Brazilian Atlantic Rain Forest, but also in Amazonia and Central America, in other biomes. The circumscription and internal relationships of Galipeinae are currently under study by the authors. One of the recognized genera of Galipeinae is Almeidea (Fig 1A and 1B), characterized as shrubs or treelets from forest understorey, with 1-foliolate, alternate leaves, a (sub)terminal thyrse and pink, lilac or white (in A. albiflora, recently described [20]) flowers with five sta- mens, of which up to three can be modified into staminodes (lacking anthers). Five species, all endemics to Brazilian Atlantic Rain Forest, were recognized in the last revision of the genus [21], but since then, collections of A. rubra from Bolivia have been identified. Almeidea are morphologically similar to Conchocarpus (Fig 1D and 1E) but differ from the latter by their free petals (vs. coherent or connate in Conchocarpus) and, with few exceptions, by their pink or lilac color (vs. creamy-white). Conchocarpus are delimited by a combination of character states, with some of these states also present in other genera of Galipeinae [16], in- cluding in Almeidea. The similarity between Almeidea and Conchocarpus is reflected in the fre- quent misidentification of herbarium specimens in a vegetative state or fruiting condition. In a previous phylogenetic analysis of the subtribe using the plastid markers trnL-trnF and rps16 [22], the single species of Almeidea and the three species of Conchocarpus that were included appeared in the same clade together with a few other genera from Galipeinae. The objective of the present study is, therefore, to perform the first phylogenetic analysis to be focused on the Galipeinae and to include a greater number of species of these two genera as well as of related genera that have not yet been included in such a study, primarily to test the PLOS ONE | DOI:10.1371/journal.pone.0125650 May 7, 2015 2/20 Almeidea Belongs to Conchocarpus and a First Analysis of Galipeinae Fig 1. Photos of some species used in this study. A. Inflorescence of Almeidea rubra. B. Flowers of Almeidea albiflora. C. Flower of Andreadoxa flava.D. Habit of Conchocarpus macrophyllus. E. Flower of Conchocarpus macrophyllus. F. Flower of Erythrochiton brasiliensis. G. Flower of Esenbeckia grandiflora H. Flower of Galipea jasminiflora. All by Milton Groppo except F (by Cláudio N. Fraga) and H (by Flávio A. Bonatti). doi:10.1371/journal.pone.0125650.g001 PLOS ONE | DOI:10.1371/journal.pone.0125650 May 7, 2015 3/20 Almeidea Belongs to Conchocarpus and a First Analysis of Galipeinae monophyly of Conchocarpus and its relationship with Almeidea. The results obtained will pro- vide a framework for taxonomic decisions about the genera cited above, a base to support dis- cussion of putative morphological synapomorphies of the groups, and a first step to a better understanding of the phylogeny of the Galipeinae and the Galipeeae. Material and Methods Taxa studied All taxa